Clonality of HTLV-2 in natural infection.

Human T-lymphotropic virus type 1 (HTLV-1) and type 2 (HTLV-2) both cause lifelong persistent infections, but differ in their clinical outcomes. HTLV-1 infection causes a chronic or acute T-lymphocytic malignancy in up to 5% of infected individuals whereas HTLV-2 has not been unequivocally linked to a T-cell malignancy. Virus-driven clonal proliferation of infected cells both in vitro and in vivo has been demonstrated in HTLV-1 infection. However, T-cell clonality in HTLV-2 infection has not been rigorously characterized. In this study we used a high-throughput approach in conjunction with flow cytometric sorting to identify and quantify HTLV-2-infected T-cell clones in 28 individuals with natural infection. We show that while genome-wide integration site preferences in vivo were similar to those found in HTLV-1 infection, expansion of HTLV-2-infected clones did not demonstrate the same significant association with the genomic environment of the integrated provirus. The proviral load in HTLV-2 is almost confined to CD8+ T-cells and is composed of a small number of often highly expanded clones. The HTLV-2 load correlated significantly with the degree of dispersion of the clone frequency distribution, which was highly stable over ∼8 years. These results suggest that there are significant differences in the selection forces that control the clonal expansion of virus-infected cells in HTLV-1 and HTLV-2 infection. In addition, our data demonstrate that strong virus-driven proliferation per se does not predispose to malignant transformation in oncoretroviral infections.

Alpha interferon restricts human T-lymphotropic virus type 1 and 2 de novo infection through PKR activation.

Type I interferon (IFN-I) inhibits the replication of different viruses. However, the effect of IFN-I on the human T-lymphotropic virus type 1 (HTLV-1) viral cycle is controversial. Here, we investigated the consequences of IFN-α addition for different steps of HTLV-1 and HTLV-2 infection. We first show that alpha interferon (IFN-α) efficiently impairs HTLV-1 and HTLV-2 de novo infection in a T cell line and in primary lymphocytes. Using pseudotyped viruses expressing HTLV-1 envelope, we then show that cell-free infection is insensitive to IFN-α, demonstrating that the cytokine does not affect the early stages of the viral cycle. In contrast, intracellular levels of Gag, Env, or Tax protein are affected by IFN-α treatment in T cells, primary lymphocytes, or 293T cells transfected with HTLV-1 or HTLV-2 molecular clones, demonstrating that IFN-α acts during the late stages of infection. We show that IFN-α does not affect Tax-mediated transcription and acts at a posttranscriptional level. Using either small interfering RNA (siRNA) directed against PKR or a PKR inhibitor, we demonstrate that PKR, whose expression is induced by interferon, plays a major role in IFN-α-induced HTLV-1/2 inhibition. These results indicate that IFN-α has a strong repressive effect on the HTLV-1 and HTLV-2 viral cycle during de novo infection of cells that are natural targets of the viruses.

HTLV-2 Tax immortalizes human CD4+ memory T lymphocytes by oncogenic activation and dysregulation of autophagy.

Human T cell leukemia virus type 1 and type 2 (HTLV-1 and -2) are two closely related retroviruses with the former causing adult T cell leukemia. HTLV-2 infection is prevalent among intravenous drug users, and the viral genome encodes the viral transactivator Tax, which is highly homologous to the transforming protein Tax from HTLV-1. However, the link between HTLV-2 infection and leukemia has not been established. In the present study, we evaluated the activity of HTLV-2 Tax in promoting aberrant proliferation of human CD4 T lymphocytes. Tax2 efficiently immortalized CD4(+) memory T lymphocytes with a CD3/TCRαß/CD4/CD25/CD45RO/CD69 immunophenotype, promoted constitutive activation of PI3K/Akt, IκB kinase/NF-κB, mitogen-activated protein kinase, and STAT3, and it also increased the level of Mcl-1. Disruption of these oncogenic pathways led to growth retardation and apoptotic cell death of the Tax2-established T cell lines. We further found that Tax2 induced autophagy by interacting with the autophagy molecule complex containing Beclin1 and PI3K class III to form the LC3(+) autophagosome. Tax2-mediated autophagy promoted survival and proliferation of the immortalized T cells. The present study demonstrated the oncogenic properties of Tax2 in human T cells and also implicated Tax2 in serving as a molecular tool to generate distinct T cell subtype lines.

Intracellular localization and cellular factors interaction of HTLV-1 and HTLV-2 Tax proteins: similarities and functional differences.

Human T-lymphotropic viruses type 1 (HTLV-1) and type 2 (HTLV-2) present very similar genomic structures but HTLV-1 is more pathogenic than HTLV-2. Is this difference due to their transactivating Tax proteins, Tax-1 and Tax-2, which are responsible for viral and cellular gene activation? Do Tax-1 and Tax-2 differ in their cellular localization and in their interaction pattern with cellular factors? In this review, we summarize Tax-1 and Tax-2 structural and phenotypic properties, their interaction with factors involved in signal transduction and their localization-related behavior within the cell. Special attention will be given to the distinctions between Tax-1 and Tax-2 that likely play an important role in their transactivation activity.

Postgenomic up-regulation of CCL3L1 expression in HTLV-2-infected persons curtails HIV-1 replication.

Leukocytes of persons coinfected with HTLV-2 and HIV-1 secrete chemokines that prevent CCR5-dependent (R5) HIV-1 infection of CD4+ T cells and macrophages, with HTLV-2-induced MIP-1alpha as dominant HIV-1 inhibitory molecule. Two nonallelic genes code for CCL3 and CCL3L1 isoforms of MIP-1alpha, and the population-specific copy number of CCL3L1 exerts a profound effect on HIV-1 susceptibility and disease progression. Here, we demonstrate that CCL3L1 is secreted spontaneously by leukocytes of HTLV-2-infected persons and superinduced when cells of HTLV-2/HIV-1 multiply exposed-uninfected seronegative (MEU) persons were stimulated with HIV-1 Env peptides. The CCL3L1 median copy number in MEU, HTLV-2/HIV-1-coinfected long-term nonprogressors (LTNPs) and HIV-1-monoinfected LTNPs were 1, 2, and 3, respectively. An increased CCL3L1/CCL3 mRNA ratio versus PHA-activated healthy leukocytes was observed in both HIV-1-monoinfected LTNPs and in HTLV-2/HIV-1(MEU) subjects. An additional potential correlate of HTLV-2 infection was a rapid and persistent leukocyte secretion of GM-CSF and IFN-gamma, 2 cytokines endowed with CCR5 down-regulation capacity. This study confirms a crucial protective role of CCL3L1 from both HIV infection and disease progression, highlighting a previously not described functional up-regulation of this chemokine variant in both HIV-positive and -negative persons infected with HTLV-2.

Neuropilin-1 is involved in human T-cell lymphotropic virus type 1 entry.

Human T-cell lymphotropic virus type 1 (HTLV-1) is transmitted through a viral synapse and enters target cells via interaction with the glucose transporter GLUT1. Here, we show that Neuropilin-1 (NRP1), the receptor for semaphorin-3A and VEGF-A165 and a member of the immune synapse, is also a physical and functional partner of HTLV-1 envelope (Env) proteins. HTLV-1 Env and NRP1 complexes are formed in cotransfected cells, and endogenous NRP1 contributes to the binding of HTLV-1 Env to target cells. NRP1 overexpression increases HTLV-1 Env-dependent syncytium formation. Moreover, overexpression of NRP1 increases both HTLV-1 and HTLV-2 Env-dependent infection, whereas down-regulation of endogenous NRP1 has the opposite effect. Finally, overexpressed GLUT1, NRP1, and Env form ternary complexes in transfected cells, and endogenous NRP1 and GLUT1 colocalize in membrane junctions formed between uninfected and HTLV-1-infected T cells. These data show that NRP1 is involved in HTLV-1 and HTLV-2 entry, suggesting that the HTLV receptor has a multicomponent nature.

Spontaneous production of C-C chemokines by individuals infected with human T lymphotropic virus type II (HTLV-II) alone and HTLV-II/HIV-1 coinfected individuals.

To investigate the immunological features of human T lymphotropic virus type II (HTLV-II) infection and specific mechanisms whereby HTLV-II might influence the progression of HIV-1 disease in coinfected individuals, we have analyzed the production of the C-C chemokines RANTES and macrophage inflammatory proteins 1alpha and 1alpha (MIP-1alpha and MIP-1beta) by PBMCs from HTLV-II-infected and HTLV-II/HIV-1-coinfected individuals. We observed spontaneous production of significant levels of MIP-1alpha and -1beta and, to a lesser extent, RANTES, from individuals infected with HTLV-II alone or with concomitant HIV-1 infection. Spontaneous C-C chemokine production was not observed in PBMCs from uninfected or HIV-1-infected individuals. Although HTLV-II is known to preferentially infect CD8+ lymphocytes in vivo, we observed that whereas RANTES was produced exclusively by the CD8+-enriched fraction, MIP-1alpha and -1beta were produced by both the CD8+-enriched and CD8+-depleted fractions of HTLV-II-infected PBMCs. RT-PCR demonstrated active expression of the HTLV-II regulatory protein Tax in the infected CD8+ T lymphocyte population, and it was further shown that Tax transactivates the promoters of MIP-1beta and RANTES. Therefore, it appears that HTLV-II stimulates the production of C-C chemokines both directly at a transcriptional level via the viral transactivator Tax and also indirectly. Although the HTLV-II-infected individuals in this study are all virtually asymptomatic, they certainly display an abnormal immune phenotype. Moreover, our findings suggest that HTLV-II, via chemokine production, would be expected to alter the progression of HIV-1 infection in coinfected individuals.

Comparative performances of an HTLV-I/II EIA and other serologic and PCR assays on samples from persons at risk for HTLV-II infection.

BACKGROUND: HTLV-I and HTLV-II are related exogenous pathogenic human retroviruses. Until recently, ELISAs based on HTLV-I antigens have been used to screen for the presence of HTLV-I or -II antibodies. The HTLV-I-based assays have not been as sensitive in detecting antibodies to HTLV-II as in detecting antibodies to HTLV-I. The Abbott HTLV-I/HTLV-II ELISA uses a combination of HTLV-I and HTLV-II antigens to detect antibodies to the whole HTLV group. The performance of this ELISA was compared to that of several HTLV-I-based serologic assays and an HTLV-II PCR assay in cohorts of South American Indians and New York City IV drug users (IVDUs) in whom HTLV-II is endemic. STUDY DESIGN AND METHODS: Sera from 429 South American Indians and New York City IVDUs were evaluated for HTLV antibodies by the use of three ELISAs (rgp21-enhanced HTLV-I/II, Cambridge Biotech; Vironostika HTLV-I/II, Organon Teknika; and HTLV-I/HTLV-II, Abbott), and a Western blot (WB) assay. Peripheral blood leukocyte DNA from each person was analyzed for HTLV-I and HTLV-II pol DNA via PCR. The HTLV-II PCR-positive samples were further subtyped via cloning and sequencing and/or oligomer restriction. RESULTS: Two hundred four samples (48%) were positive for HTLV-II by serologic and/or PCR assays. All of the positive samples from the Indians and approximately one-third of the positive samples from the IVDUs were of the HTLV-IIB subtype. Comparative analyses indicate that the sensitivity and specificity of the various assays were: PCR, 98 and 100 percent; Abbott HTLV-I/HTLV-II, 78 and 95 percent; Cambridge Biotech HTLV-I/II, 76 and 96 percent; Vironostika HTLV-I/II, 71 and 98 percent; and WB, 73 and 100 percent, respectively. CONCLUSION: There were no significant differences among the sensitivities and specificities of the HTLV-I/II ELISAs (p values, 0.056-0.438). The WB and PCR assays were much more specific than the other serologic assays (p

Restricción étnica y geográfica de la infección causada por el virus HTLV-II y su asociación con el polimorfismo del complejo mayor de histocompatibilidad en tres subpoblaciones del Caribe colombiano

Las infecciones causadas por los virus HTLV-I y II parecen tener una distribución geográfica y étnica entre amerindios y otras poblaciones nativas en el mundo. En Colombia existe un foco endémico entre afrocolombianos del litoral Pacífico, no así en la costa del Caribe. Objetivos: Establecer una asociación entre la diversidad genética del MHC y la infección causada por el virus HTLV-II en tres grupos étnicos representativos del Caribe colombiano. Encontrar la prevalencia de la infección causada por los virus HTLV-II en una muestra representativa de estos respectivos grupos étnicos. Métodos: Se colectó suero de 157 indios wayuu, 840 mestizos y 580 afro-colombianos, buscando la presencia de anticuerpos anti HTLV-I/II usando dos técnicas diferentes: Una pueba de aglutinación pasiva (PA, Serodia, Fujirobio, Tokio) y una prueba de micro Elisa (Murex). La oligotipificación de los antígenos HLA clase-II fue realizada mediante PCR-SSOP, siguiendo el protocolo de la 12 IHWSC y se realizó en un total de 41 muestras indigenas wayuu, 61 mestizos y 100 afrocolombianos. Resultados: las muestras pertenecientes a los mestizos y a los afrocolombianos resultaron negativas; once sueros de la población wayuu fueron repetidamente reactivas con PA y micro Elisa, reconfirmadas usando una prueba de Western Blot (HTLV-Blot, 2.4 Gene Laboratory), obteniéndose una seroprevalencia al HTLV-II de 7 por ciento entre los wayuu. Los alelos con mayor frecuencia en los wayuu fueron DRb1*0411 (46 por ciento), y DQb1*0302 (83 por ciento). Analizando los haplotipos en los 11 wayuu seropositivos encontramos en todos ellos la expresión del haplotipo HLA DRb1*0411-DQB1*0302, (p<005). Ninguno de los 75 mestizos y los 100 afro-colombianos lo expresaron. Conclusiones: Los resultados obtenidos sugiren una restricción genética de la suceptibilidad a ser infectado por este virus y podrían explicar la distribución geográfica y étnica de la infección causada por HTLV-II en estos grupos estudiados.

HTLV-II risk factors in Native Americans in Florida.

Earlier virologic studies established that human T-cell lymphotropic virus type II (HTLV-II) is the predominant retrovirus type found among Seminole Indians in southern Florida. We studied 46 members of the Seminole tribe living on 3 reservations to determine the risk factors for HTLV-II and to investigate disease association with the virus. The donors' plasma samples were evaluated with the enzyme-linked immunosorbent and Western blot assays. DNA extracted from their peripheral blood mononuclear cells were analyzed by type-specific polymerase chain reaction amplification and detection of the HTLV pol gene using the primer pair SK110/SK111, and the probes SK112 or SK188. One of 46 (2%) subjects was identified as HTLV-I positive and 11 (24%) were identified as HTLV-II positive. Restriction fragment length polymorphism and sequence analyses indicated that all of the HTLV-II strains were subtype b. Mitochondrial DNA analyses indicated that all of the HTLV-II-positive subjects had an Amerindian haplotype. HTLV-II was more prevalent in Indians who were >45 years of age or female, had multiple sex partners or had received a blood transfusion. However, only the latter risk factor was statistically significant. Three of the HTLV-II-positive Indians demonstrated signs and symptoms of an ataxic neuropathy. The data support that HTLV-IIb is endemic among the Seminoles and that they will be a key population for further virologic studies.

HTLV-I and HTLV-II virus expression increase with HIV-1 coinfection.

Coinfections with HIV-1 and HTLV-I or HTLV-II have been associated with unique immunophenotypes and an increased risk for development of neurodegenerative conditions. These findings may result from an increased HTLV-I or II viral burden in dually infected individuals. To investigate this possibility, HTLV-I/II tax/rex messenger RNA and viral antigen expression in peripheral blood mononuclear cells (PBMCs) were measured in 37 HTLV-I- or HTLV-II-infected subjects with or without HIV-1 coinfection. Tax/rex messenger RNA was detected in 14 of 24 PBMC samples from dually infected subjects, compared with only 1 of 13 PBMC samples from singly infected subjects (58% versus 7%; p < .003). The reverse transcription-polymerase chain reaction (RT-PCR) assay correlated with HTLV-I/II viral antigen detection in PBMC cultures but not with HIV-1 viral load levels in plasma. These findings may provide clues regarding the pathophysiologic consequences of HIV/HTLV-I and HIV/HTLV-II coinfections.

Endemic infection with HTLV-IIB in Venezuelan Indians: molecular characterization.

The peripheral blood of 41 Yaruro and Guahibo Indians from Venezuela was examined for HTLV antibodies and DNA. Twenty-five samples (61%) were found to be infected with HTLV-IIB. The sensitivities of the serologic and DNA polymerase chain reaction (PCR) analyses were 80% and 96%, respectively. Epidemiologic studies supported both sexual and perinatal transmission of the virus. Sequence analyses of the HTLV-IIB strains from these Indians indicate that they are unique relative to HTLV-II detected in other groups of humans. HTLV-IIB-G2 isolated from a Guahibo Indian is the most divergent HTLV-IIB strain relative to the prototype HTLV-II NRA.

Role of the genetic background of rats in infection by HTLV-I and HTLV-II and in the development of associated diseases.

Three aspects of the rat model of HTLV-I/II infection were investigated. (i) The efficacy of HTLV-I-transformed rat cell lines in infecting different strains of rats: WKY and Lewis HTLV-I-transformed cell lines were injected into adult WKY, Lewis and Brown Norway rats, representing syngeneic and allogeneic combinations. The HTLV-I provirus was not detected in peripheral-blood mononuclear cells (PBMC) from these rats 18 weeks after inoculation, showing that HTLV-I-transformed rat cells are not suitable for virus challenge in vaccination experiments. Rats inoculated with Lewis HTLV-I-transformed cells produced an antibody response to HTLV-I, which was higher in allogeneic (WKY and Brown Norway) than in syngeneic rats. (ii) The susceptibility of rats to HTLV-II infection: After human HTLV-II-producing cells (MO) were injected into adult WKY rats, the HTLV-II provirus was detected in PBMC 12 weeks later. Sequencing of a portion of this provirus confirmed its identity with the HTLV-II from MO cells. (iii) The role of MHC haplotype in susceptibility to neurological disease in rats inoculated as newborns with HTLV-I: The hypothesis that the RT-Ik haplotype confers susceptibility was tested by inoculating newborn OKA (RT-Ik), WKY (RT-Il), Lewis (RT-Il) and Fischer 344 (RT-I lvl) rats with human HTLV-I-producing cells (MT-2). Eighteen months later, only the WKY rats showed histological abnormality of the spinal cord, without clinical paralysis. Fischer 344 rats developed cutaneous tumors and OKA rats mammary tumors. The HTLV-I provirus was not detected in these tumors.

Human T-cell leukemia viruses: epidemiology, biology, and pathogenesis.

The human T-cell lymphotropic viruses type I and type II are closely related human retroviruses that have similar biological properties, genetic organization and tropism for T lymphocytes. Along with the simian T-cell lymphoma virus type I, they define the group of retroviruses known as the primate T-cell leukemia/lymphoma viruses. Initially identified in 1980, the human T-cell lymphotropic virus type I has been implicated as the etiologic agent of adult T-cell leukemia/lymphoma and of a degenerative neurologic disorder known as tropical spastic paraparesis or human T-cell lymphotropic virus type I-associated myelopathy. The intriguing link between human T-cell lymphotropic virus type, T-cell malignancy, and a totally unrelated and non-overlapping neurological disorder suggests divergent and unique pathogenetic mechanisms. This review will address the epidemiology, molecular biology, and pathogenesis of human T-cell leukemia viruses.

Molecular analysis of HTLV-I and HTLV-II isolates from Italian blood donors, intravenous drug users and prisoners.

Using molecular methods three or five major variants of HTLV-I have been identified; moreover two subtypes of HTLV-II defined as HTLV-IIa and HTLV-IIb with six variants within each of these groups have been described. In the present study we analysed proviral DNA obtained from the peripheral blood mononuclear cells (PBMCs) of a significant group of Italian intravenous drug users (IVDUs), prison inmates and blood donors (BDs) who were HTLV antibody positive. Restriction fragment length polymorphism (RFLP) of amplified LTR region with ApaI, NdeI, DraI, SacI and MaeIII endonucleases was used to define the HTLV-I subtypes, while the different variants of HTLV-IIa and -IIb were defined by RFLP of the LTR region with the AvaII, BglI, SauI, XhoI and BanII endonucleases. The four HTLV-I isolated from BDs were characterized as C type. All the 11 HTLV-II detected in the IVDUs were HTLV-IIb4, while among the prisoners one HTLV-IIb5 and five HTLV-IIb4 were found. Interestingly, in the BDs group two HTLV-IIa0 and one HTLV-IIb4 were detected. It should also be noted that 82% of the IVDUs and 50% of the prisoners were coinfected with HIV, while all the BDs were HIV negative. These data indicate that HTLV-IIb4 is the predominant genotype in Italian IVDUs and prisoners, while the significant variability observed in the BD HTLV-II isolates could be due to the different source of infection among this group.

Nucleotide sequence and restriction fragment-length polymorphism analysis of human T-cell lymphotropic virus type II (HTLV-II) in southern Europe: evidence for the HTLV-IIa and HTLV-IIb subtypes.

Human T-cell lymphotropic virus type II (HTLV-II) has been subtyped into two major groups, IIa and IIb, according to molecular studies involving env gene sequencing. Subsequently, this retrovirus was further subclassified by examining the long terminal repeat (LTR), the most divergent genomic region. Sequence analysis and restriction fragment-length polymorphism (RFLP) applied to the LTR region identified either four or five groups within the IIa subtype (depending on the restriction enzyme sets used) and six within the IIb subtype. In this study, we analyzed the LTR sequences of 29 samples obtained from HTLV-II-infected individuals living in Spain and Italy, which included 24 injecting drug users (IDUs), three blood donors, and two subjects at risk for HIV/HTLV infection. Sequence analysis and phylogenetic analysis of 720 base pairs of the LTR performed in 10 Spanish samples showed that all of these samples belonged to IIb subtype, with a divergence of 7.5% and 1.66% compared with MoT (IIa) and NRA/G12 (IIb) isolates, respectively. RFLP analysis demonstrated the presence of the IIb 4-subtype restriction pattern in 26 samples, a IIb5-subtype pattern in one Italian IDU, and a IIa0-subtype pattern in two Italian samples (blood donors), according to W.M. Switzer's nomenclature. This is the first report of the presence of IIb5 in Southern Europe and IIa0 among Italian blood donors. RFLP correlated with nucleotide sequence and phylogenetic data obtained in this study, demonstrating the ability of the RFLP method to predict the phylogroup of HTLV-II-infected samples.

Highly endemic human T-lymphotropic virus type II (HTLV-II) infection in a Venezuelan Guahibo Amerindian group.

Sera from 166 Guahibo Indians (55% of the population) living in southwest Venezuela were screened by enzyme-linked immunoassay for antibodies to human T-cell lymphotropic virus (HTLV) I and II. Positive samples were confirmed by immunofluorescence and Western blot. Forty-one Guahibos (24.8%) were found to be seropositive. Polymerase chain reaction (PCR) analysis of proviral DNA in mononuclear cell lysates revealed the virus to be HTLV-II. Prevalence increased with age, and sexual contact with HTLV-II-seropositive partners was identified as a risk factor for infection. PCR amplification of a region of the pol gene, utilizing the primer pair SK110/SK111, with subsequent digestion of the 140-base-pair amplification products with HinfI and MseI restriction enzymes, showed an HTLV-II subtype-b restriction pattern in all cases. These data suggest that the substrain infecting this Guahibo community belongs to the b subtype, the most frequent among Paleo-Amerindian populations.